Cellular signaling by the fibroblast growth factor (FGF) family of ligands (FGF1-10 and FGF16-23) plays essential roles in mammalian development and metabolism. FGFs execute their diverse activities by binding, dimerizing and activating FGF receptor tyrosine kinases (FGFRs) in a heparan sulfate (HS)-dependent fashion. Mammalian FGFR genes (FGFR1-4) encode single pass transmembrane receptors composed of an extracellular ligand binding region, and a cytoplasmic region that harbors the conserved tyrosine kinase domain. The endocrine acting FGFs also require the presence of Klotho proteins in their target tissues. Upon activation the cytoplasmic domain of FGFR associates with intracellular signaling molecules in either phosphorylation dependent or independent fashion to initiate distinct intracellular pathways. Reflective of the pleiotropic roles of FGF signaling in human biology, deregulated FGF signaling leads to a wide array of human diseases, including skeletal, olfactory/reproductive syndromes, hearing loss, phosphate wasting disorders, and cancer. The recent discovery of the endocrine-acting FGF19 subfamily's critical roles in maintaining bile acid, glucose, lipid, and phosphate homeostasis has sparked renewed interest in the therapeutic potential of FGFs. Consequently, there is a major impetus to comprehend the molecular mechanisms of FGF signaling as the results of such studies should facilitate the development of novel therapeutics for the treatment of a variety of human diseases. The specific aims are: I. Explore the role of klotho co-receptors in the regulation of FGF-FGFR binding specificity. II. Dissect the requirement for A-loop tyrosine phosphorylation in the hyperactivation of FGFR tyrosine kinase by pathogenic gain-of-function mutations. III. Determine the order by which intracellular signaling molecules are recruited to the cytoplasmic region of FGFR, and investigate the existence of cooperativity between recruitment events. Recombinant protein expression, Surface Plasmon Resonance (SPR) spectroscopy, steady-state kinetics analysis, and mass spectrometry will be used to accomplish the Specific Aims of this proposal. These studies should lead to the first demonstration that the ligand binding specificity of an RTK can be altered by tissue specific cell surface co-receptors thus providing a new layer of regulatory mechanism in maintaining signaling specificity of RTKs. Moreover, our studies should enhance our understanding of how the extracellularly occurring ligand-receptor binding and dimerization events can be appropriately decoded into selective activation of intracellular signaling pathways. Finally, our data should show how pathogenic mutations activate FGFRs and aid in discovery of new therapeutics to interfere with unregulated activation of FGFR and possibly other RTKs. PUBLIC HEALTH RELEVANCE: Fibroblast growth factor receptors (FGFRs) play essential roles in human development and metabolism. The overall aim of this application is to understand how the activity of FGFRs is regulated and to unveil the mechanisms by which naturally occurring pathogenic mutations corrupt receptor function to give rise to various human pathologies including skeletal, olfactory/reproductive syndromes, and cancer. Thus the results of our studies should facilitate the development of novel therapeutics for the treatment of a variety of human diseases.